Utilizing DNA transfection analysis with the continuous NIH/3T3 cell line as the assay system, we have observed that the DNA of a primary human diffuse B cell lymphoma induced an unusual transformed focus upon transfection in NIH/3T3 cells. The transforming gene was serially transmissible, conferred the neoplastic phenotype to NIH/3T3 cells and appeared to be larger than 20 kbp by analysis of transfectants for conserved human DNA sequences. From a cosmid recombinant DNA library of a third-cycle transfectant, overlapping clones spanning 80 kbp of cellular DNA were isolated. One clone, which contained a 45-kbp insert comprised entirely of human sequences, was shown to be biologically active, with a specific transforming activity of 650 focus-forming units/pmole. By restriction mapping and hybridization analysis, this human transforming gene was shown to be unrelated to any previously reported oncogene. In a small series of methylcholanthrene (MCA)-induced fibrosarcoma-derived cell lines, we earlier had shown the specific activation of K-ras in 50% of the tumor cells analyzed. In more extensive studies, we analyzed by transfection assay, DNAs of MCA-induced fibrosarcomas in BALB/c and NIH Swiss mice for their ability to transform NIH/3T3 cells. An activated K-ras gene was detected in 50% of the tumor DNA analyzed. We noticed a relationship between the frequency of activated K-ras in MCA-induced tumors and the latency period as well as the growth capability of the tumors themselves. The K-ras-positive cells also demonstrated strikingly faster growth of tumor cells in vivo as compared with the K-ras-negative cells. We also analyzed, by transfection experiments, DNAs of thymic lymphomas induced in RFJ mice by MCA and DNAs of liver tumors induced in mice by diethylnitrosomine (DEN). We found that K-ras was specifically activated in the MCA-induced lymphomas, while H-ras was the transforming gene found in about 40% of DEN-induced liver tumors.